Abstract A critical function for all living organisms is the ability to move when needed. These movements--intracellular trafficking, cell division, muscle contraction, and cell motility-- are driven by molecular machines that exert an amazing amount of force considering that they are only a few nanometers across. Given the variety of motor proteins in the cell, a key question is how motors cooperate and compete while moving cargoes and applying forces. An emerging paradigm is the notion of specialized motors, or motors that are fine-tuned to perform a specific function. Despite the importance of these motor proteins, relatively little is known about their individual adaptations and how these relate to the motility patterns found in the cell. This work focuses on myosin-6 and myosin-10 and their unique forms of cellular regulation. Myosin-6 plays essential roles in organelle morphology, cell morphology, cytokinesis, autophagy, and endocytosis, while myosin-10 delivers essential cargoes such as integrins, cadherins and netrin receptors to filopodia at the leading edge of the cell. Both are overexpressed in tumors, owing to their roles in membrane trafficking, cell migration, and metastasis. The work will develop new approaches to control myosin-6 and isolate its activity in cells. One of the main approaches will be to sequester myosin-6 with light, making cells that have an optically switchable Snell's waltzer (myosin-6 null) phenotype. Experiments are designed to identify when myosin-6 acts as a passenger, an anchor, or a transporter in the cells, and if direct handoff from one cargo adaptor to another is required for function. The proposed work will also investigate how cargo binding and myosin quaternary structure tune myosin-10's motility. An integrated approach is developed, combining structural studies with functional reconstitution and single molecule motility assays. This proposal will test the hypotheses that cargo and extracellular ligand binding are both required for myosin-10 activation, and that cargo can steer myosin-10 from one type of actin network to another. Completion of this study will yield a comprehensive view of how cytoskeletal motor proteins are activated and regulated for distinct tasks in the cell. Motor protein regulation is a process of fundamental biological importance, but is poorly understood. This work will direct future efforts to understand activation in multiple contexts.